Use of indisulam for the preparation of antiviral medicaments

Abstract

The application provides application of Indisulam in preparation of antiviral drugs, and relates to the technical field of medicines.In the application, it is first disclosed that Indisulam can be applied in preparation of antiviral drugs; the molecular glue Indisulam can target RBM39 protein, affect splicing of an influenza virus M gene, and then inhibit replication of the influenza virus, thereby providing a new functional component for antiviral drugs, especially for drugs against influenza A virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the application of Indisulam in the preparation of an antiviral drug. BACKGROUND

[0002] Influenza A virus (IAV) is a respiratory virus that causes zoonosis, has strong infectivity, and can easily cause seasonal epidemics or even global pandemics, posing a serious threat to public health safety. According to the mechanism of action, anti-influenza virus drugs mainly include ion channel inhibitors such as amantadine, neuraminidase inhibitors such as oseltamivir, and influenza virus acid polymer PA inhibitors such as marabivir. Due to the continuous mutation and recombination of the influenza virus genome, the problem of drug resistance of antiviral drugs targeting viral components is becoming increasingly serious. For example, amantadine is no longer used to treat influenza, and the phenomenon of oseltamivir and marabivir resistance is also increasingly prominent. Therefore, there is an urgent need in the art to develop a new type of anti-influenza virus drug targeting the host.

[0003] Pre-mRNA alternative splicing is an indispensable part of the life cycle of influenza virus, which is completed in dependence on the participation of multiple host factors, so the host factors involved in influenza virus mRNA alternative splicing are extremely attractive targets for the development of new antiviral drugs. The RNA-binding Motif 39 (RBM39) protein contains a conserved RNA recognition motif (RRM), which participates in the selection of splicing sites by binding to RNA molecules, and recruits spliceosome components to complete mRNA splicing. Indisulam and dCeMM1 are a kind of molecular glue degrader based on the ubiquitination-proteasome system, which promotes the degradation of RBM39 protein by binding RBM39 with DCAF15-CRL4 E3 ubiquitin ligase. Indisulam is a potential antitumor drug for acute myeloid leukemia, malignant melanoma and colorectal cancer, and has entered the second phase of clinical experimental research. The role of RBM39 in influenza virus mRNA splicing and the potential of molecular glue degrader targeting RBM39 in anti-influenza virus are still unknown and need to be clarified. SUMMARY

[0004] To solve the above problems, the present application first found that Indisulam can be applied to the preparation of an antiviral drug, which has an inhibitory effect on influenza A virus.

[0005] In one aspect, the present application provides the application of Indisulam in the preparation of an antiviral drug.

[0006] Indisulam (E 7070) is a carbonic anhydrase inhibitor, which is known to have anticancer activity in the prior art. The CAS# of Indisulam is 165668-41-7.

[0007] Further, the virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0008] Further, the virus is influenza A virus.

[0009] It is first proved in the present application that the EC50 of Indisulam to influenza A virus is 0.1018 μM, the CC50 is 187.1 μM, and the selectivity index is 1852.47, which has a good effect on anti-influenza, especially anti-influenza A virus.

[0010] Further, the Indisulam inhibits the virus by regulating the level of RBM39.

[0011] The regulation of the level of RBM39 is down-regulation of the level of RBM39, and the down-regulation of the level of RBM39 leads to splicing error of mRNA of influenza A virus, specifically, affects the splicing of M gene, so as to realize the anti-virus effect.

[0012] In another aspect, the present application also provides an anti-virus drug, which includes Indisulam.

[0013] The person skilled in the art can select the use concentration of Indisulam according to the actual situation, which is not specifically limited here.

[0014] In a preferred embodiment, the use concentration of Indisulam can be 0.0625-4 μM.

[0015] Further, the virus includes one or more of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0016] Further, the virus is influenza A virus.

[0017] Further, the drug further includes a pharmaceutically acceptable excipient.

[0018] The present application can also add adjuvants, which can be appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integration agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0019] The drug of the present application can be prepared by a general method, in which one or more diluents or carriers can be added. The carrier and specific dosage form of the drug can be selected by a person skilled in the art according to the needs.

[0020] Further, the dosage form of the drug can be pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions and / or suspensions.

[0021] The present application has the following beneficial effects:

[0022] It is first proved in the present application that RBM39 can serve as a splicing pivot and become a new target of "host-targeted antiviral drugs", and the effect of resisting influenza virus is achieved by degrading RBM39.

[0023] It is also first disclosed in the present application that Indisulam can be applied to prepare antiviral drugs, and the molecular glue Indisulam can inhibit influenza virus replication by targeting RBM39 protein and affecting the splicing of influenza virus M gene. A new functional component is provided for antiviral drugs, especially for anti-influenza A virus drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0025] Figure 1 It is a statistical graph of RBM39 expression level for Example 1;

[0026] Figure 2 It is a Western Blot detection graph for knocking down RBM39 protein to inhibit influenza virus replication;

[0027] Figure 3 It is a Western Blot detection graph for degrading RBM39 to inhibit influenza virus replication;

[0028] Figure 4 It is a statistical graph of virus growth titer inhibited by dCeMM1;

[0029] Figure 5 Graph of relative values of IAV growth titers for comparison of Indisulam and dCeMMl inhibition;

[0030] Figure 6 Graph of Western Blot detection of IAV splicing proteins for comparison of Indisulam and dCeMMl inhibition;

[0031] Figure 7 Graph of EC50 for Indisulam;

[0032] Figure 8 Graph of CC50 for Indisulam;

[0033] Figure 9 Imaging of IAV mRNA splicing in RBM39 knockdown cell lines;

[0034] Figure 10 Graph of IAV mRNA splicing inhibition by Indisulam;

[0035] Figure 11 Imaging of IAV mRNA splicing in RBM39 degradation. DETAILED DESCRIPTION

[0036] In order to more clearly illustrate the overall teachings of the present application, the following detailed description is provided with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without one or more of these specific details. In other instances, well-known features have not been described in detail to avoid obscuring aspects of the present application.

[0037] It is to be understood that the following detailed description is exemplary of the present application and intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains.

[0038] Before further description of the application, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as to the scope of the present application.

[0039] In the following examples, the reagents or instruments used are not specified by the manufacturer unless otherwise specified, and are all conventional products that can be obtained by commercial purchase. In the examples, the specific conditions are not specified, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0040] The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercially available products, and the specific operations are carried out according to the kit instructions.

[0041] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all use conventional molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology and related fields of conventional technology, and can be carried out according to the molecular cloning experiment guide (Molecular Cloning: A Laboratory Manual (Fourth Edition)).

[0042] In addition, the "water" described in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and any feasible water that can be used in the field.

[0043] In the following examples, if no other special instructions are given, vt% is used, that is, volume percentage.

[0044] The culture media involved in the following examples are as follows:

[0045] The cell culture medium is DMEM medium containing 10% FBS.

[0046] RBM39 is an RNA binding protein that binds pre-mRNA by recognizing specific RNA motifs, recruits spliceosome proteins, and plays an important role in pre-mRNA maturation. The M2 protein of influenza virus is an essential ion channel protein of influenza virus, which plays a crucial role in the process of influenza virus invading host cells and maturation and release of progeny virions. M2 protein is translated from M2 mRNA produced by splicing of M mRNA. Influenza virus lacks splicing machinery and relies on host factors to complete its mRNA splicing. In this application, it is first verified that RBM39 can be used as a splicing hub and can become a new target of "host-targeted antiviral drugs", and the effect of antiviral is achieved by degrading RBM39.

[0047] Example 1 Construction and verification of RBM39 knockdown cell line

[0048] 1.1 Design of sgRNA and construction of expression vector

[0049] (1) The NCBI number of RBM39 mRNA is NC_000020.11. The guide RNA (sgRNA) sequence targeting RBM39 is designed as follows:

[0050] sgRNA1: 5'-GTGCCATCCGAGGAAAGATT-3' (SEQ ID No. 1);

[0051] sgRNA2: 5'-CTCAAGAAGTCGAGATCGAA-3' (SEQ ID No. 2).

[0052] (2) The sgRNA primer synthesized by the above company is subjected to phosphorylation treatment, and the reaction system is shown in Table 1.

[0053] Table 1

[0054]

[0055] The mixed reaction solution is placed in a PCR instrument to anneal to obtain double-stranded sgRNA. The annealing program is as follows: 37℃, 30min; 95℃, 5min; 95℃-25℃, -5℃ / s; 25℃, 1min, and the double-stranded after annealing is taken out after the program ends, and the primer is diluted by water at a ratio of 1:1000.

[0056] (3) Use BsmB I to cut the pLentiCRISPR V2 vector, and react at 55℃ for 4h.

[0057] (4) The target fragment (about 15000 bp in size) in step (3) is recovered by nucleic acid gel electrophoresis, and the vector is connected with the double-stranded sgRNA after dilution and reannealing in step (2), and the reaction system is shown in Table 2.

[0058] Table 2

[0059]

[0060] (5) The connection product obtained in step (4) is transformed into STBL3 competent cells, and the LB plate containing ampicillin is coated and cultured at 37℃, and a single colony is picked and sent for sequencing verification. The plasmid pLentiCRISPR V2 is obtained.

[0061] 1.2 Construction of RBM39 knockdown cell line

[0062] 16-24h before transfection, 293T cells are seeded in a 6-well plate at 2x10 6Cells were seeded at a concentration of 5.4 μg / dish in 6 cm dishes. Transfection was performed after the cell density reached 70%-80%. The transfection ratio was pLentiCRISPR V2:psPAX2:pMD2.G = 4:3:1, with 5.4 μg plasmid and 13.5 μL PEI. Forty-eight hours post-transfection, the cell supernatant was collected and filtered through a 0.22 μM sterile filter. A2-well plates containing A549 cells seeded 18 hours prior were then used at a density of 1.6 × 10⁶ cells / well. 5 Cells were cultured at a density of 1 cell / mL. The residual culture medium in the wells was removed, and the cells were washed twice with PBS. 500 μL of supernatant was added to the medium. At the same time, a group of WT cells without supernatant was left as a control. After 48-72 h of infection, the medium was replaced with puromycin (final concentration of 1 μg / mL) and cultured until all cells in the WT group died, thus obtaining RBM39-depletion cells.

[0063] 1.3 Detection of RBM39 expression in RBM39-depletion cells using qRT-PCR.

[0064] (1) Design primers for qRT-PCR (see Table 3 for specific primer sequences) to detect RBM39 gene expression.

[0065] Table 3

[0066]

[0067] (2) The control (WT) A549 cells and RBM39-depletion cells were seeded into 12-well plates and cultured in an incubator for 15-20 h. After the cells were confluent, the culture medium in the corresponding wells was discarded, the cells were washed twice with PBS, and 500 μL of Trizol was added.

[0068] (3) Extract RNA and reverse transcribe the extracted RNA into cDNA using a reverse transcription kit.

[0069] (4) Prepare qRT-PCR reaction solution. The system is shown in Table 4. Use RBM39-F / R primers from Table 3.

[0070] Table 4

[0071]

[0072] (5) Add the reaction solution from step (4) into a qRT-PCR-specific well plate (three replicates per group), cover the well plate with a special membrane, and centrifuge briefly to bring all the liquid to the bottom of the well plate. Place it in a dedicated qRT-PCR instrument and run it according to the instrument's default program.

[0073] (6) After processing the obtained data, GraphPadPrism 10.1.2 was used to plot the graph, and the result is as follows. Figure 1 As shown, sgAAVS1 refers to the control group, with RBM39 expression at 100%; sgRBM39_1 refers to the group using sgRNA1 to knock down RBM39, with RBM39 expression at 60%; and sgRBM39_2 refers to the group using sgRNA2 to knock down RBM39, with RBM39 expression at 25%. This demonstrates that transducing sgRNA1 and sgRNA2 to reduce RBM39 expression yields the RBM39 knockdown cell line.

[0074] Example 2: Assessment of Viral Proliferation in RBM39 Knockdown Cell Lines

[0075] The viral replication of the RBM39 knockdown cell line (RBM39-depletion cells) constructed in Example 1 (taking the RBM39 knockdown group using sgRNA2 as an example) was tested. The specific testing method is as follows:

[0076] The control (WT) A549 cells and RBM39-depletion cells constructed in Example 1 were inoculated into a 12-well plate and cultured in an incubator for 20 h. WSN virus was infected at 0.01 MOI. 500 μL of virus infection solution containing the required virus was added to each well, and adsorbed at room temperature for 1 h. The virus infection solution containing the original virus was aspirated, washed twice with PBS, and 1 mL of virus-free virus maintenance solution (containing 0.5% fetal bovine serum, 0.5 μg / mL TPCK-trypsin in DMEM) was added. At 9 h, 24 h, and 48 h after virus infection, the culture medium in the corresponding well was discarded, washed twice with PBS, and the cells were lysed with 70 μL of RIPA lysis solution, incubated on ice for 30 min, then centrifuged at 12000 rpm for 15 min, and 65 μL of lysis solution was added to 13 μL of protein loading buffer, boiled at 98°C for 8 min, cooled to room temperature, and then centrifuged briefly for SDS-PAGE electrophoresis. After electrophoresis, the protein samples in the gel were transferred to a nitrocellulose membrane at a current of 280 mA for 90 min. After blocking with 5% skim milk for 1 h, the primary antibodies RBM39 (67420-1-Ig, proteintech), β-actin (66009-1-Ig, proteintech), M1 (GTX636675, GeneTex), NA (GTX125974, GeneTex), and NP (GTX125989, GeneTex) were diluted 1:1000 and incubated at room temperature for 2 h, then washed with TBST (4 times, 5 min each time), and the fluorescent secondary antibodies Rb800 (926-32213, LI-COR) and Mu680 (926-68073, LI-COR) were diluted 1:10000 and incubated at room temperature for 30 min, then washed with TBST (4 times, 5 min each time), and developed using a dual-color infrared laser imaging system. The results are shown in Figure 2 Figure 2, and the replication of influenza virus in the RBM39-depletion cell line was significantly weakened. This result demonstrates that reducing the expression of RBM39 can achieve the effect of inhibiting the replication of influenza virus.

[0077] Example 3 Detection of the Anti-IAV Effect of RBM39 Degradation

[0078] 3.1 Detection of the Anti-IAV Effect of RBM39 Degradation by Indisualm Using Western Blot Method

[0079] A549 cells were seeded into 12-well plates about 15-20 h before, different drug concentrations (0, 0.1, 0.3, 1 μM) of Indisualm (control group is DMSO) were added directly into the cell culture medium. Shake gently, incubate at 37°C, 5% CO2 incubator for 1 h, then infect WSN virus. The A549 cells pretreated with drugs were infected with 0.001 MOI. After 1 h of infection, the medium was changed, and the virus maintenance solution contained the corresponding dose of DMSO or Indisulam. The cell culture supernatant was taken 48 h after virus infection for plaque assay to detect virus titer, and the cells were lysed with RIPA lysis buffer, and the protein sample was collected, and the influenza virus replication was detected by Western Blot method. The subsequent steps are the same as Example 2.

[0080] The results are shown in Figure 3 As the dose of Indisulam increases, the influenza virus replication is significantly weakened.

[0081] 3.2 Detection of dCeMM1 degradation of RBM39 anti-IAV effect by plaque assay

[0082] (1) A549 cells were seeded into 12-well plates about 15-20 h before, different drug concentrations (0, 0.625, 0.125, 0.5 μM) of dCeMM1 (control group is DMSO) were added directly into the cell culture medium. Shake gently, incubate at 37°C, 5% CO2 incubator for 1 h, then infect WSN virus. The A549 cells pretreated with drugs were infected with 0.001 MOI. After 1 h of infection, the medium was changed, and the virus maintenance solution contained the corresponding dose of dCeMM1 / DMSO. The cell culture supernatant was taken 72 h after virus infection for plaque assay to detect virus titer.

[0083] (2) Prepare MDCK cells one day in advance, plate 12-well plates (about 1.2 x 10 5 cells per well), cell density is about 70%-80%. Infect the cells with virus at 10 -3 -10 -5The virus was serially diluted (three replicates per group). 450 μL of virus solution was prepared for each dilution. A Vortex vortex mixer was used during dilution to ensure thorough mixing. The culture medium for MDCK cells was aspirated, and the cells were washed twice with PBS. As needed, 400 mL of virus dilution was added to each well, gently mixed, and incubated for 2 h. 2% low-melting-point agarose (LMT) was melted in a microwave oven and incubated in a 37°C water bath. 2% LMT was diluted 1:1 with preheated virus maintenance medium (containing 1% BSA and 0.5 μg / mL TPCK-trypsin in 2×DMEM) to a final concentration of 1%. After virus incubation, the cells were washed once with PBS to remove unadsorbed virus particles. Approximately 1.2 mL of the diluted 1% agarose was added to each well. The cells were incubated at room temperature for 30 min until the agarose completely solidified, then inverted and placed in an incubator. Results were observed after 72 h. Fix with 4% tissue fixative at room temperature for 10 min, remove agar, stain with crystal violet for 5 min, rinse with tap water, observe the results, and calculate the viral titer in the cell supernatant.

[0084] The calculation formula is:

[0085]

[0086] The obtained data was processed using GraphPadPrism 10.1.2 software, and the results are as follows. Figure 4 As shown, the influenza virus titer in A549 cells treated with dCeMM1 was significantly lower than that in control cells treated with DMSO.

[0087] 3.3 Comparison of the anti-IAV effects of dCeMM1 and Indisualm

[0088] According to Example 3.3.1, protein samples were collected for Western blotting, and the supernatant was used to determine the viral titer. The results are as follows: Figure 5 and Figure 6 As shown, Figure 5 The results demonstrated that at the same concentration (0.125 μM), Indisulam had a stronger inhibitory effect on influenza virus compared to dCeMM1. Figure 6 The results showed that Indisulam could inhibit the expression of influenza virus M2 protein at a lower concentration than dCeMM1, indicating that Indisulam exerts a stronger anti-influenza virus effect.

[0089] Example 4: Evaluating the potential of Indisulam in anti-IAV applications

[0090] 4.1 Detection of the anti-IAV effect of RBM39 degradation using the pitting test

[0091] The supernatant was collected and the viral titer was determined in the experiment of Example 3.1. The concentration of Indisulam was 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 μM, respectively. The specific operation process was the same as 3.2 (2). The results showed that the titer of influenza virus in A549 cells with Indisulam was significantly lower than that in control cells (added with DMSO). The obtained data was processed by GraphPad Prism 10.1.2 software, as shown in Figure 7 The EC50 of Indisulam on IAV was 0.1018 μM.

[0092] 4.2 Detection of Indisulam cytotoxicity by CCK-8

[0093] A549 cells were seeded in 96-well plates (10000 cells / well), and the plates were pre-cultured in the incubator for 15-20 h. 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 μM drugs (3 replicates for each group) were added to the 96-well plates, and the plates were placed in the incubator for continuous culture for 24 h. Then 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the incubator for 1 h. The absorbance value of each well at 450 nm was determined by using a microplate reader (SpectraMax S). The obtained data was processed by GraphPad Prism 10.1.2 software, as shown in Figure 8 The CC50 of Indisulam was 187.1 μM.

[0094] The calculation formula involved above is:

[0095]

[0096] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of control wells (containing cells, culture medium, CCK-8 solution, without drug); Ab: absorbance of blank wells (containing culture medium, CCK-8 solution, without cells and drug).

[0097] 4.3 Calculation of selectivity index to evaluate the potential of Indisulam in the application of anti-IAV

[0098] The calculation formula of selectivity index showed that the drug selectivity index of Indisulam on influenza virus was 1852.47.

[0099] The calculation formula of selectivity index is:

[0100]

[0101] The EC50 of favipiravir against influenza virus is known to be 341 nM, and the CC50 is 100 μM. The selectivity index is 293. As can be seen, compared with the existing art common antiviral drugs, Indisulam has better antiviral effect on influenza virus, and it is proved in this embodiment that the method is non-toxic to cells and has high safety.

[0102] Example 5 Detection of IAV mRNA splicing in RBM39 knockdown cell lines

[0103] (1) The primers for semi-qRT-PCR (see Table 5 for specific sequences) were designed to detect the expression and splicing of influenza virus M / NS genes.

[0104] Table 5

[0105]

[0106] (2) The constructed control (WT) A549 cells and RBM39-depletion cells (sgRBM39#1 for RBM39 knockdown using sgRNA1, sgRBM39#2 for RBM39 knockdown using sgRNA2, and sgControl for blank control) were inoculated into 12-well plates and cultured in an incubator for 15-20 h. WSN was infected at 1 MOI. After 1 h of infection, the culture medium was replaced with virus maintenance liquid containing the corresponding dose of Indisulam. After 5 h of infection, the culture medium was discarded, washed twice with PBS, and 500 μL of Trizol was added.

[0107] (3) RNA was extracted using the Trizol method, and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (no Radom was added in the second step of reverse transcription).

[0108] (4) The cDNA reverse transcribed in the previous step was subjected to PCR (primers: rtM-F1, rtM-F2, rtM-R, rtNS-F1, rtNS-F2, rtNS-R in Table 1), and the system is shown in Table 6.

[0109] Table 6

[0110]

[0111] (5) The mixture of step (4) was placed in a PCR instrument, and the M gene amplification program is shown in Table 7, and the NS gene amplification program is shown in Table 8.

[0112] Table 7

[0113]

[0114] Table 8

[0115]

[0116] (6) Prepare 2% agarose gel, take 15 μL of PCR product for nucleic acid electrophoresis, 80V for 90 min. After electrophoresis, develop in ChemiDoc imaging system. As shown in Figure 9 , RBM39 mainly affects the splicing of influenza virus M gene to achieve the effect of inhibiting the virus.

[0117] Example 6 Detection of IAV mRNA splicing under RBM39 degradation

[0118] 6.1 qRT-PCR detection of M / NS gene expression and splicing

[0119] (1) Design qRT-PCR primers (see Table 9 for specific primer sequences) to detect influenza virus M / NS gene expression and splicing.

[0120] Table 9

[0121]

[0122] (2) A549 cells were seeded in 12-well plates and cultured in an incubator for 15-20 h. 1 h before infection, 1 μM Indisulam was added to the wells (DMSO for the control group) to infect WSN at 1 MOI. Replace with virus maintenance solution containing the corresponding dose of DMSO or Indisulam 1 h after infection. Discard the culture medium in the corresponding wells at 4 h, 6 h, and 8 h after infection, wash twice with PBS, and add 500 μL Trizol.

[0123] (3) Perform RT-qPCR according to the procedure in Example 1, step 1.3, using primers qM1-F, qM1-R, qM2-F, qM2-R, qNS1-F, qNS1-R, qNS2-F, and qNS2-R.

[0124] (4) After processing the obtained data, use GraphPad Prism 10.1.2 for plotting, and the results are shown in Figure 10 , which prove that Indisulam mainly affects the splicing of influenza virus M gene to inhibit the replication of influenza virus.

[0125] 6.2 semi-qRT-PCR detection of M / NS gene expression and splicing

[0126] (1) The primer sequences are the same as in Example 5 (1).

[0127] (2) A549 cells were seeded in 12-well plates and incubated in an incubator for 15-20 h. DMSO or (1 μM) Indisulam was added to the wells 1 h before infection, and A549 cells were infected at 1 MOI. Virus maintenance solution containing the corresponding dose of DMSO or Indisulam was added 1 h after infection. The medium was discarded 8 h after infection, and the cells were washed twice with PBS, and 500 μL Trizol was added.

[0128] (3) RT-PCR was performed according to Example 5(3)-(5).

[0129] (4) A 2% nucleic acid gel was prepared, and 15 μL of the PCR product was run on the nucleic acid electrophoresis for 80 V for 90 min. After electrophoresis, the nucleic acid gel was developed in a ChemiDoc imaging system. The results, as shown in Figure 11 indicated that Indisulam mainly affects the splicing of the influenza virus M gene, thereby inhibiting the replication of the influenza virus.

[0130] The above description is merely illustrative of the embodiments of the present application and not intended to limit the present application. Alternatives and variations of the present application are apparent to those of ordinary skill in the art without departing from the spirit and principles of the present application. Accordingly, the scope of the present application should be determined by reasonable interpretation of the appended claims and all technical equivalents thereof.

Claims

1. Use of indisulam for the preparation of an antiviral medicament, characterized in that, The virus is an influenza A virus. The virus is an influenza A virus. The virus is an influenza A virus. The virus

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